I have a question about westernblots. Please help - (Jun/18/2007 )

I used nitrocelluous memberane to transfer my western gel. After blocking I was trying to probe the memberane and I accidently left at room temp. over night. Then I found that my primary/secondary probing is not working. Could anybody tell me why this is happening? Thanks a lot

-ppmm-

QUOTE (ppmm @ Jun 19 2007, 01:11 PM)

I used nitrocelluous memberane to transfer my western gel. After blocking I was trying to probe the memberane and I accidently left at room temp. over night. Then I found that my primary/secondary probing is not working. Could anybody tell me why this is happening? Thanks a lot

Well, overnight should be fine at RT.

Did you include a positive control on your membrane? Have you used this antibody and antigen before and seen that it works? Do you know the appropriate dilution to use for your serum?

Scott.

-ScottC-

Thanks Scott. I did include a positive control on my blot. I've used the entibody before and it worked very well.

-ppmm-

RT over night should have enhanced the recognition of your protein by your antibody, not abolished it.

Are you sure that the proteins were on the membrane? Have your positive control worked?

-Madrius-

QUOTE (Madrius @ Jun 20 2007, 11:26 PM)

RT over night should have enhanced the recognition of your protein by your antibody, not abolished it.

Are you sure that the proteins were on the membrane? Have your positive control worked?

Are you using milk for blocking and Ab incubation? If so and your milk is not so good, there might be problem with o/n incubation. In that case, your membrane will be coated with milk and you will have a blank WB.